Inflammatory bowel disease (IBD), consisting of ulcerative colitis (UC) and Crohn's disease, is a source of substantial morbidity for 1.4 million people affected in the USA, and it can progress to colon cancer. It is difficult to treat, with cosly immunotherapies only inducing remission in less than half of cases. We have been focusing on the role of the semi-essential amino acid, L-arginine (L-Arg) as a complementary and alternative medicine. We have demonstrated mechanisms for beneficial effects of L-Arg in vitro and in colitis. Cationic amino acid transporter 2 (CAT2), the inducible transporter of L-Arg, and uptake of L-Arg are upregulated in murine colitis, and oral L-Arg supplementation is effective as a treatment for epithelial injury and inflammation induced by dextran sulfate sodium (DSS), a model that mimics UC. CAT2 expression, L-Arg uptake, and L-Arg levels are all decreased in UC tissues. CAT2-/- mice exhibit marked exacerbation of DSS colitis, and colon tumorigenesis in the azoxymethane (AOM)-DSS model of colitis-associated carcinoma (CAC). CAT2-/- mice do not respond to L-Arg supplementation in the DSS colitis model, suggesting that L-Arg alone as a therapy for human UC may not be sufficient to overcome its impaired transport into tissues. Colonic epithelial restitution in a wound repair model is dependent on CAT2 and this is due to utilization of L-Arg by arginase that generates L-ornithine (L-Orn), which can be metabolized by either ornithine decarboxylase (ODC) to generate polyamines, or ornithine aminotransferase (OAT) to generate L-proline (L-Pro). While inhibition of arginase or knockdown of arginase 1 (Arg1) prevented restitution, this could be completely restored, in the presence of L- Arg, when either L-Orn or L-Pro was added. Knockdown of OAT, but not ODC, prevented beneficial effects of L-Arg on restitution, implicating OAT in the maintenance of epithelial function. Additionally, ODC+/- mice exhibit improvement in DSS colitis, with increased tissue macrophage NO production, and enhanced regulatory T cell and macrophage responses, indicating a deleterious role for polyamines in this model. We hypothesize that benefits of L-Arg in colitis and colitis-associated tumorigenesis depend on CAT2 and downstream effectors to improve epithelial restitution, innate immune function, and adaptive immunity. In our Specific Aims we will determine if: 1) exacerbation of DSS colitis due to deletion of CAT2 and loss of L-Arg availability that mimics human UC is due to an epithelial or macrophage defect and if this is ameliorated by supplementation of L-Orn or L-Pro in combination with L-Arg; 2.) improvement in DSS colitis in ODC+/- mice is due to an epithelial or macrophage effect and if it results from enhanced L-Arg availability for iNOS and/or OAT; 3.) accelerated tumorigenesis with CAT2 deletion is due to an epithelial or macrophage defect and if this process can be beneficially modulated by downregulation of ODC or supplementation of L-Orn or L-Pro in combination with L- Arg. Through investigation of epithelial and immune function, these studies seek to provide new macronutrient- based strategies for treatment of IBD and prevention of colitis-associated dysplasia and carcinoma.